Magnesium-based coatings for nanocrystals

ABSTRACT

Semiconductor nanocrystal compositions comprising magnesium containing shells and methods of preparing them are described. The compositions provide strong emission in the blue and green wavelengths as well as chemical and photostability that have not been achieved with conventional shell materials.

This application is a Continuation of U.S. Non-Provisional applicationSer. No. 14/865,264, filed on Sep. 25, 2015, which is a Divisional ofU.S. Non-Provisional application Ser. No. 12/602,493, filed Aug. 12,2010, which is a 371 of PCT/US2008/065425, filed May 30, 2008, whichclaims priority to U.S. Provisional Application Ser. No. 60/941,211,filed May 31, 2007, which is expressly incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The invention relates to semiconductor nanocrystals comprising a coreand a magnesium-containing layer.

DESCRIPTION OF RELATED ART

Semiconductor nanocrystals are important new materials that have a widevariety of applications. Of the many unique properties of thesematerials, the photophysical characteristics may be the most useful.Specifically, these materials can display intense luminescent emissionthat is particle size-dependent and particle composition-dependent, canhave an extremely narrow luminescence bandwidth, can be environmentallyinsensitive, and are resistant to photobleaching under intensive lightsources. Emissions can be efficiently excited with electromagneticradiation having a shorter wavelength than the highest energy emitter inthe material. These properties allow for the use of semiconductornanocrystals as, for example, ultra-sensitive luminescent reporters ofbiological states and processes in highly multiplexed systems.

Nanocrystal cores have been broadly studied and improvements insynthesis have lead to the optimization of key physiochemical propertiesresulting in nanocrystal cores with uniform size distributions andintense, narrow emission bands following photo-excitation. However,nanocrystal cores alone lack sufficiently intense or stable emissionintensities for most applications, and nanocrystal cores areparticularly sensitive to their environment; for example, the aqueousenvironment required for many biological applications can lead to thecomplete destruction of the luminescence of nanocrystal cores. Thusmethods to photostabilize nanocrystal cores (e.g., protect theirluminescent properties) and make them stable and useful in aqueous mediaare of great interest for biological applications.

The ability to coat nanocrystal cores has been an area of much research,and coating nanocrystal cores with an inorganic shell to form“core/shell nanocrystals”, has resulted in improved emission intensity,chemical and photochemical stability, reduced self-quenchingcharacteristics, stability in a variety of environments, and the like.The impact of coating nanocrystal cores with an inorganic shell onunderlying luminescence energies is not well understood and is generallycontrolled based on a small set of criteria such as, for example, thechoice of the coating material and the density and thickness of theshell. Optionally, an organic or other overcoat that is selected toprovide compatibility with a dispersion medium may surround the shell.

An inorganic shell is generally thought to passivate the outermostsurface of a core nanocrystal thereby reducing or eliminating thesurface energy states associated with the core and insulating the corefrom the outside environment. This can reduce or eliminate thenonradiative loss of excitons from the core to the environment.Photochemical degradation may, therefore, be reduced, and emissionsefficiency and stability may be improved by coating a core with aninorganic shell.

The choice of shell material largely depends on the core material. Forexample, the shell material may generally have a wider band gap than thecore, and the shell may be chosen to have an atomic spacing and latticestructure that closely match those of the core material. Core/shellnanocrystals having a CdX core wherein X is S, Se, or Te coated with aYZ shell where Y is Cd or Zn, and Z is S, Se, or Te are commonlyproduced and used and have been shown to have good emissionscharacteristics and stability. This may largely be due to the YZ coatingmaterial's band-gap energy which spans that of the core relativelysymmetrically. ‘Symmetry’ as used in this sense means that the widerbandgap of the shell material fully encompasses the narrower bandgap ofthe core material and extends both above the high end of the corematerial's bandgap and below the low end of the core material's bandgap.For example, FIGS. 1 and 2 show the band gap alignments for CdSe coreswith CdS or ZnS shells. Both CdS and ZnS span the CdSe band gap, but ZnSspans CdSe more symmetrically, extending both above the high end of theCdSe bandgap and below the low end of the CdSe bandgap. Therefore, aCdSe/ZnS core/shell nanocrystals may have better properties than aCdSe/CdS nanocrystal since the symmetry of the band gap alignment maybetter insulate the core from the surrounding environment.

It must also be recognized that the bandgaps for each of these materialsis a value measured for the bulk material rather than for a nanocrystalcomprised of that material. The bandgap for a material is known tochange as the size of the sample of the material changes—that is anunderlying basis for the uniquely valuable fluorescence properties ofnanocrystals, for example. It is not well understood how the bandgaps ofvarious materials or mixtures of materials are affected as the sampleapproaches nanocrystal dimensions. In particular, a thin layer of asemiconductor material forming a shell on a nanocrystal may not havebandgap properties that are predictable relative to its bulk properties.Thus, while the bulk material properties can be useful to explain whycertain pairs of materials work well, they may not be reliable forpredicting which pairings will work well.

One limitation of CdSe-based core/shell nanocrystals is that the blueemitting particles have lower extinction coefficients than red emittingparticles. This is due to the fact that emission wavelength are tuned bychanging the CdSe core particle size. Several researchers have shownthat by utilizing alloy cores (e.g., CdSSe or ZnCdSe) one can tune thewavelength by adjusting the elemental composition rather than size anddecouple emission color from extinction coefficient. One can alsoutilize a semiconductor material with a larger bulk band gap such thatthe largest nanocrystals emit in the blue/green (e.g. ZnSe).

Nanocrystal cores have limited utility without passivation with aninorganic shell. ZnSe or ZnCdSe cores, while having advantageousfluorescence properties, have proven to be much more difficult to coatand passivate than CdSe cores. The failure to passivate these cores isprimarily due to their inherently high conduction band energy. Asillustrated in FIGS. 3 and 4, the band gap energy of ZnSe core does notalign well with either CdS or ZnS. Even though ZnS may span the coreband gap better, it is not symmetrical—it does not extend much above thehigh end of the ZnSe bandgap. In fact, the band gap misalignment of ZnSeand ZnS semiconductor material is so severe when these materials arereduced into a nanocrystal size, that even when coated with severalmonolayers of ZnS the emissions intensity of ZnSe/ZnS is rapidly lostwhen exposed to oxygen.

Thus, there exists a need for materials and methods suitable to form apassivating shell over nanocrystal cores with high conduction bands(e.g. ZnSe cores).

Many patents and publications exist relating to semiconductornanocrystals. The following is a small selection of those available inthe field relating to magnesium-containing nanocrystals.

A publication by Hee Son, D. et al. (Science 306: 1009-1012 (2004))describes cationic exchange reactions in ionic nanocrystals. CdSenanocrystals were reacted with Ag⁺ ions to yield Ag₂Se nanocrystals. Theauthors reported surprising speed and reversibility of the reaction atroom temperature.

U.S. Pat. No. 5,537,000 (issued Jul. 16, 1996) describes monolayers ofsemiconductor nanocrystals. A list of suitable semiconductor materialsis presented, including MgS, MgSe, and MgTe.

U.S. Pat. No. 6,306,610 (issued Oct. 23, 2001) offers fluorescentsemiconductor nanocrystals associated to a compound. In discussing thenanocrystals, a long list of core and shell materials is disclosedincluding MgS, MgSe, and MgTe.

U.S. Pat. No. 6,379,622 (issued Apr. 30, 2002) describes a device fordetecting the presence of analyte in a sample. The device contains abinding substrate, a labeled analogue, a dye bound to the bindingsubstrate, a first reference containing quantum dots that emit light ata second wavelength when irradiated, an analyte-permeable membrane, anda void volume. In discussing the quantum dots, a long list ofsemiconductor materials are listed, including MgS, MgSe, and MgTe.

U.S. Pat. No. 6,444,143 (issued Sep. 3, 2002), U.S. Pat. No. 6,251,303(issued Jun. 26, 2001), and U.S. Pat. No. 6,319,426 (issued Nov. 20,2001), and U.S. Pat. No. 6,426,513 (issued Jul. 30, 2002) offersemiconductor nanocrystals coated with various materials. Overcoatinglayers containing ZnS, GaN, or magnesium chalcogenides such as MgS, MgSeand MgTe are suggested.

U.S. Pat. No. 6,500,622 (issued Dec. 31, 2002) describes the use ofsemiconductor nanocrystals in bead-based nucleic acid assays. Indiscussing the nanocrystal shells, a material that has a bandgap energyin the ultraviolet is suggested, for example ZnS, GaN, and magnesiumchalcogenides such as MgS, MgSe, and MgTe.

U.S. Pat. No. 6,602,671 (issued Aug. 5, 2003) offers semiconductornanocrystals for fluid dynamics, microfluidics, identification ofobjects, encoding combinatorial libraries, and genomics applications. Indescribing the nanocrystals, overcoating layers containing ZnS, GaN, ormagnesium chalcogenides such as MgS, MgSe and MgTe are suggested.

U.S. Pat. No. 6,630,307 (issued Oct. 7, 2003) provides methods ofdetecting target analytes in samples. In discussing shelled nanocrystalsfor use in the methods, a material that has a bandgap energy in theultraviolet is suggested, for example ZnS, GaN, and magnesiumchalcogenides such as MgS, MgSe, and MgTe.

U.S. Pat. No. 6,645,444 (issued Nov. 11, 2003) describes methods for thepreparation of metal nanocrystals. Metal ions are complexed with organicligands, and reduced to form nanocrystals. Magnesium is among the longlist of metallic elements suggested as suitable for formingnanocrystals.

U.S. Pat. No. 6,653,080 (issued Nov. 23, 2003) describes loop probehybridization assays for polynucleotide analysis using semiconductornanocrystals. In discussing the nanocrystal shells, a material that hasa bandgap energy in the ultraviolet is suggested, for example ZnS, GaN,and magnesium chalcogenides such as MgS, MgSe, and MgTe.

U.S. Pat. No. 6,423,551 (issued Jul. 23, 2002) and U.S. Pat. No.6,699,723 (issued Mar. 2, 2004) offer core-shell nanocrystals withattached linking groups. A list of semiconductor materials includes MgS,MgSe, and MgTe.

U.S. Pat. No. 6,921,496 (issued Jul. 26, 2005) suggests ionic conjugatesof fusion proteins and semiconductor nanocrystals. The nanocrystals canhave the formula MX; where M is cadmium, zinc, magnesium, mercury,aluminum, gallium, indium or thallium.

U.S. Pat. No. 6,734,420 (issued May 11, 2004) offers a spectral bar codesystem using semiconductor nanocrystals. In discussing the nanocrystalshells, a material that has a bandgap energy in the ultraviolet issuggested, for example ZnS, GaN, and magnesium chalcogenides such asMgS. MgSe. and MgTe.

U.S. Pat. No. 6,759,235 (issued Jul. 6, 2004) offers two-dimensionalspectral imaging systems using semiconductor nanocrystals. In discussingthe nanocrystal shells, a material that has a bandgap energy in theultraviolet is suggested, for example ZnS, GaN, and magnesiumchalcogenides such as MgS, MgSe, and MgTe.

U.S. Pat. No. 6,774,361 (issued) describes methods of identifying itemslabeled with semiconductor nanocrystals. In discussing the nanocrystalshells, a material that has a bandgap energy in the ultraviolet issuggested, for example ZnS, GaN, and magnesium chalcogenides such asMgS, MgSe, and MgTe.

U.S. Pat. No. 6,819,692 (issued Nov. 16, 2004) describes a laser andgain media. The gain media contains a plurality of semiconductornanocrystals of formula MX; where M is cadmium, zinc, magnesium,mercury, aluminum, gallium, indium or thallium.

U.S. Pat. No. 6,821,337 (issued Nov. 23, 2004) and U.S. Pat. No.7,138,098 (issued Nov. 21, 2006) offer a method of synthesizing ananocrystal by combining a metal-containing non-organometallic compound,a coordinating solvent, and a chalcogen source to form a nanocrystal.Magnesium is one of several metals listed.

U.S. Pat. No. 6,838,243 (issued Jan. 4, 2005) describes polynucleotideanalysis using generic capture sequences. Semiconductor nanocrystals areused as at least one label in the methods. In discussing shellednanocrystals, a material that has a bandgap energy in the ultraviolet issuggested, for example ZnS, GaN, and magnesium chalcogenides such asMgS, MgSe, and MgTe.

U.S. Pat. No. 7,068,898 (issued Jun. 27, 2006) describes composites of amatrix and nanostructures. In discussing shelled nanostructures, amaterial that has a bandgap energy in the ultraviolet is suggested, forexample ZnS, GaN, and magnesium chalcogenides such as MgS, MgSe, andMgTe.

U.S. Pat. No. 7,079,241 (issued Jul. 18, 2006) describes spatialpositioning of spectrally labeled beads. In discussing shellednanocrystals, a material that has a bandgap energy in the ultraviolet issuggested, for example ZnS, GaN, and magnesium chalcogenides such asMgS, MgSe, and MgTe.

U.S. Pat. No. 7,108,915 (issued Sep. 19, 2006) offers water-dispersiblenanocrystals prepared by applying a coating of a multiply amphipathicdispersant to the surface of a hydrophobic nanocrystal. In discussingthe nanocrystal shells, a material that has a bandgap energy in theultraviolet is suggested, for example ZnS, GaN, and magnesiumchalcogenides such as MgS, MgSe, and MgTe.

U.S. Pat. No. 7,129,048 (issued Oct. 31, 2006) describes loop probehybridization assays for polynucleotide analysis using semiconductornanocrystals. In discussing the nanocrystal shells, a material that hasa bandgap energy in the ultraviolet is suggested, for example ZnS, GaN,and magnesium chalcogenides such as MgS, MgSe, and MgTe.

U.S. Pat. No. 7,150,910 (issued Dec. 19, 2006) describes a gratinghaving a semiconductor nanocrystal layer. The nanocrystals can have theformula MX, where M is cadmium, zinc, magnesium, mercury, aluminum,gallium, indium, thallium, or mixtures thereof.

SUMMARY

Provided herein are compositions and methods that address certainsignificant limitations of conventional CdSe core-based nanocrystals. Insome embodiments, provided herein are nanocrystals comprisingZn-containing cores along with shells that successfully improve orprotect the core's luminescence properties. In some embodiments, theimproved nanocrystals provide greater luminescent intensity thanconventional CdSe core nanocrystals having the same maximum emissionwavelength. In some embodiments, the improved nanocrystals also provideenhanced photostability and/or chemical stability relative toconventional CdSe core nanocrystals. Methods for making nanocrystalshaving such improved properties are also provided, including methodsthat minimize concerns about matching lattice spacing of the shell withthe underlying core. The disclosed nanocrystals have improvedphotostability and intensity relative to conventional nanocrystalshaving CdSe cores, and they can be made across a wider color range thannanocrystals known in the art while maintaining high luminescentintensity and suitable photostability.

Because emission wavelengths of CdSe core based nanocrystals are tunedby changing the particle size, smaller particles that fall into the blueand green sections of the visible spectrum have lower extinctioncoefficients. This leads to conventional nanocrystals having blue andgreen emitting nanocrystals that have relatively weak emission comparedto the red emitting nanocrystals. Due to this limitation, alloys such asCdSSe and ZnCdSe and wider band gap semiconductors (e.g., ZnSe) havebeen explored. Although these materials address the problem of weaklyemitting blue/green CdSe cores, passivation of these alternative coreshas proven difficult. Thus, while providing brighter and more easilydetectable luminescence responses, these alternative cores havegenerally been less stable useful than those with CdSe cores, which havereceived most of the attention in this field.

Cores have limited utility compared to core/shells due to poor chemicaland photostability of unprotected cores. When a bare core nanocrystal isexcited into a state where the nanocrystal has a ‘separated’ hole andelectron, it is too reactive toward common species like oxygen. It thusrapidly gets damaged or degraded when illumination puts it into anexcited state. A shell applied over the core can be effective to protector passivate the core nanocrystal, provided it effectively insulates thecore from the external environment.

However, while suitable shell or passivation layers for use with CdSenanocrystal cores may be known, the selection and application of asuitable passivating layer on other types of nanocrystal cores can bedifficult. The passivation difficulty is believed to arise from the factthat most non-cadmium based semiconductors have high conduction bandsrelative to cadmium based semiconductors. These materials, whenphotoexcited, leave the excited electron vulnerable to oxidativequenching even when covered with passivation layer or shell materialslike ZnS and CdS that would be suitable for use on a CdSe core.

Provided herein are compositions that not only provide strong emissionin the blue and green wavelengths, but also provide greater chemicaland/or photostability than has been achieved with conventional shellmaterials (e.g., ZnS, ZnSe, CdS). Nanocrystals provided herein havingblue, green or blue-green emission bands and brightness that aresignificantly enhanced relative to conventional CdSe core nanocrystalsare therefore an aspect of the invention. Thus, in some embodiments,provided here are nanocrystals having comparable photostability to ananocrystal comprising a CdSe core and ZnS shell, for example, whileproviding more intense luminescence which makes it easier to detect.FIGS. 10, 11 and 12 herein demonstrate that nanocrystals of theinvention provide greater fluorescence intensities than a commercializedquantum dot known as Qdot 525, and that they also retain high levels ofphotostability; while the same core without the shells of the presentinvention had much lower photostability.

Additionally, provided herein are methods for incorporating magnesiuminto a previously deposited inorganic shell on a core/shell nanocrystal,and methods for using magnesium treatments to enhance the photostabilityof a nanocrystal. These methods overcome several previously unforeseendifficulties that were encountered in attempting to deposit MgX shellsdirectly on cores by traditional colloidal synthetic techniques; themethods of the invention should be applicable to the modification of anycore/shell nanocrystal.

In one aspect, provided herein is a bright nanocrystal comprising a coreand a shell, wherein the bright nanocrystal:

-   -   a) has a characteristic fluorescence emission wavelength in the        visible range,    -   b) is photostable, and    -   c) provides fluorescence intensity that is at least about twice        the fluorescence intensity of a conventional nanocrystal having        a CdSe core that is sized to have the same emission wavelength        as the bright nanocrystal.

The bright nanocrystal may have a characteristic fluorescence emissionwavelength of between about 400 nm and about 600 nm. In someembodiments, this wavelength is between 450 and 550, or between 500 and550 nm.

In some embodiments, the bright nanocrystal has a core that comprisesZn. In some embodiments, it has a shell that comprises Mg.

In another aspect, provided here are nanocrystals that include a corethat comprises Zn and a shell that comprises Mg. In some embodiments,the shell comprises both Mg and Zn.

In some embodiments, the core comprises ZnSe or ZnCdSe. In someembodiments, the core comprises or consists essentially of ZnCdSe, andthe ratio of Zn to Cd is between about 0.1 and about 0.3.

In certain embodiments of the above nanocrystals, the shell comprisesZnS. In certain embodiments, the shell comprises MgS. In someembodiments, it comprises both MgS and ZnS.

In some of the foregoing embodiments, the core of the nanocrystalconsists essentially of ZnCdSe. In other of the foregoing embodiments,the core of the nanocrystal consists essentially of ZnSe. In some ofthese embodiments, the shell of the nanocrystal consists essentially ofa mixture of MgS and ZnS. Other materials may be present on the shell insuch embodiments.

In some embodiments, the amount of MgS in the shell is sufficient toincrease the photostability of the nanocrystal relative to an otherwiseidentical nanocrystal that does not contain magnesium in its shell. Insome embodiments, the amount of MgS is sufficient to provide ananocrystal that loses less than 10% of its fluorescence intensity underirradiation conditions where an otherwise identical nanocrystal thatdoes not contain magnesium in its shell loses at least 20% of itsfluorescence intensity. In some embodiments, the amount of MgS issufficient to provide a nanocrystal that loses less than 10% of itsfluorescence intensity under irradiation conditions where an otherwiseidentical nanocrystal that does not contain magnesium in its shell losesat least 40% of its fluorescence intensity.

In some embodiments, provided herein is a nanocrystal that comprises aZn_(x)Cd_(1-x)Se core, where x is between 0.1 and 0.9, and a shellcomprising Mg_(y)Zn_(1-y)S, where y is at least about 0.1. In someembodiments, y is selected to provide enough MgS to form a monolayer onthe surface of the core of the nanocrystal. In some embodiments of thesenanocrystals that comprise a Zn_(x)Cd_(1-x)Se core, the nanocrystal hasa characteristic fluorescence maximum wavelength in the blue or greenportion of the visible spectrum. Preferably, such nanocrystals arephotostable. In some embodiments, x is greater than 0.15 or greater than0.25. In some embodiments, x is 0.5 or greater. In some embodiments, xis at least 0.7.

In another aspect, provided herein are methods to make a photostablenanocrystal, or to increase the photostability of a nanocrystal Anexemplary method comprises the steps of:

-   -   i) providing a first core/shell nanocrystal;    -   ii) dispersing the nanocrystal in a reaction mixture containing        a magnesium material; and    -   iii) heating the reaction mixture with the nanocrystal to a        temperature of at least about 120° C.;        -   to produce a photostable nanocrystal having better            photostability than the first core/shell nanocrystal.

In some embodiments, the methods provide a photostabilized nanocrystalthat loses less than 10% of its initial emission intensity underconditions where the first core/shell nanocrystal would lose 20% of itsinitial emission intensity. In some embodiments, the methods provide aphotostable nanocrystal that loses less than 10% of its initial emissionintensity under conditions where the first core/shell nanocrystal wouldlose 40% of its initial emission intensity.

In some embodiments, these methods use a reaction mixture that comprisesan alkylphosphinic acid, a trialkylphosphine, or a trialkylphosphineoxide, or a mixture of at least two of these materials. Thetrailkylphosphine is often utilized as a medium for conducting thereaction. In some embodiments, it is trioctylphosphine.

For the exemplary methods, the core of the first core/shell usedsometimes comprises ZnSe, or consists essentially of. In otherembodiments, the core of the first core/shell comprises ZnCdSe. or itconsists essentially of ZnCdSe. As used herein, ZnCdSe indicates that Znand Cd are both present, and frequently Zn represents about 15-95% ofthe metal (Zn+Cd) present, while the balance is Cd. In one embodiment,Zn represents about 50% of the metal and about 50% is Cd. In otherembodiments, the ratio of Zn to Cd is greater than 1. In otherembodiments, the ratio is about 2, and in other embodiments, the ratiois about 5 or greater than about 5.

In some embodiments, the methods disclosed herein are used to increasephotostability of a nanocrystal having an amount of Cd in the core thatis sufficient to provide a core having a largest dimension of about 6 nmand a fluorescence color in the blue or green region of the visiblespectrum. In some embodiments, the core has an amount of Cd in the corethat is sufficient to provide a core having a largest dimension of about6 nm and a fluorescence emission maximum at a wavelength above 500 nm.

In some embodiments of the foregoing methods, the shell of the firstcore/shell nanocrystal comprise ZnS.

In the above methods any suitable magnesium material can be used. Insome embodiments, a magnesium material identified herein is used. Insome embodiments, the magnesium material is a magnesiumalkylcarboxylate. In certain embodiments, it is magnesium acetate.

In some embodiments, the method uses a reaction mixture which, inaddition to a magnesium material, comprises at least one of thefollowing materials: a trialkylphosphine, a trialkylphosphine oxide, analkylamine, or an alkylphosphinic acid. An alkylphosphinic acid may bepresent in some embodiments, sometimes along with an alkylamine. In someembodiments, a trialkylphosphine is used as the medium or solvent forthe reaction. In certain embodiments, the mixture comprisestrioctylphosphine.

The methods typically include heating the nanocrystal in the reactionmixture. In some embodiments of the foregoing methods, the step ofheating the reaction mixture with the nanocrystal comprises heating themixture to a temperature between about 150° C. and about 250° C. In someembodiments, the step of heating the reaction mixture with thenanocrystal comprises heating the mixture for between about 0.5 hr andabout 4 hr.

In another aspect, the invention provides a photostable nanocrystal, ora photostabilized nanocrystal, made by the foregoing methods.

In another aspect, provided herein is a nanocrystal that comprises asemiconductor core and a shell, wherein the shell comprises Mg. Theamount of magnesium present can vary; typically, the amount of magnesiumis an amount that is sufficient to increase the photostability of thenanocrystal relative to an otherwise identical nanocrystal preparedwithout magnesium.

Other aspects and embodiments are described below.

DESCRIPTION OF THE FIGURES

The following figures form part of the present specification and areincluded to further demonstrate certain aspects of the presentinventions. The inventions may be better understood by reference to oneor more of these figures in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 shows the band gap energy diagram for CdSe versus CdSsemiconductive material. CdS has a wider bandgap than CdSe, and can thusbe used as a shell for a CdSe core, but the CdS bandgap does not extendmuch above the high end of the CdSe bandgap.

FIG. 2 shows the band gap energy diagram for CdSe versus ZnSsemiconductive material. ZnS has a much wider bandgap than CdSe, asillustrated here, and CdSe nanocrystals can be effectively protectedfrom degradation (photostabilized) by a shell of ZnS. This combinationis commonly used in commercial quantum dots.

FIG. 3 shows the band gap energy diagram of ZnSe versus CdSsemiconductive material.

FIG. 4 shows the band gap energy diagram of ZnSe versus ZnSsemiconductive material. FIGS. 3 and 4 show that CdS would not make agood shell for a ZnSe core, and ZnS may be a poor choice, also, judgingsolely from the bandgap information.

FIG. 5 shows the band gap energy diagram of ZnSe versus ZnS and MgSsemiconductive material. The bandgap comparison suggests that MgS mightmake a better shell for a ZnSe core than ZnS would.

FIG. 6 shows the emission intensity of hydrophobic ZnCdSeZnS core/shellsand Mg treated hydrophobic ZnCdSe/ZnS core/shells in hexane. Itillustrates that a Mg treatment method of the invention enhances thephotostability of a ZnCdSe core, and shows that the ZnCdSe core is notwell protected by a ZnS shell alone due to photoinstability.

FIG. 7 shows the emission intensity of water dispersible ZnCdSe/ZnScore/shells and Mg treated water dispersible ZnCdSeZnS core/shells inaqueous solvent. This is similar to FIG. 6, with a ZnCdSe core thatprovides a different emission maximum.

FIG. 8 is a drawing that depicts a ZnCdSe core overcoated with a ZnSshell first shell and a MgS shell second. This depicts a core having aZnS shell applied, followed by a MgS shell.

FIG. 9 is a drawing that depicts a ZnCdSe core overcoated with a shellthat is a mixture of Zn, Mg and S. This shell could be homogeneous, orit could contain a gradient having a Zn/Mg ratio that either increasesor decreases when moving outward from the core through the shell layer.

FIG. 10 shows the relative emission intensity of two streptavidinconjugates titrated against immobilized biotin. Qdot 525 is a popularcommercial quantum dot having an emission maximum at 525 nm, used as astandard. The ZnCdSe/ZnMgS is a nanocrystal of the invention having acore containing mainly Zn and a shell that contains Mg, and has asubstantially higher luminescence intensity than the commercial standardemitting at the same maximal wavelength.

FIG. 11 shows the relative emission intensity of two goat anti-rabbitconjugates titrated against immobilized rabbit antibody. Qdot 525 is apopular commercial quantum dot having an emission maximum at 525 nm,used as a standard. The ZnCdSe/ZnMgS is a nanocrystal of the inventionhaving a core containing mainly Zn and a shell that contains Mg, and hasa substantially higher luminescence intensity than the commercialstandard emitting at the same maximal wavelength.

FIG. 12 shows the relative emission intensity from fixed HEp-2 cellsincubated with rabbit anti-Ki67 antibody followed by incubation withgoat anti rabbit quantum dot conjugates. The relative emission intensityis shown for two excitation wavelengths-480 nm was used because it is awidely available excitation wavelength because it is used forfluorescein dyes, while 425 nm was used because it is optimal for theZnCdSe core. Note that the ZnCdSe/ZnMgS nanocrystal of the invention hasmuch higher fluorescence emission than the Qdot 525 standard at bothwavelengths.

FIG. 13 shows the effect on emission wavelength of doping differentamounts of cadmium into ZnSe cores. Increasing the amount of Cd used inthree successive preparations demonstrated that using more Cd usedmaking the Cd-modified cores (presumably producing cores having anincreased amount of Cd) caused the wavelength maximum for emission toincrease.

FIG. 14 shows the particle diameters measured by transmission electronmicroscopy of ZnSe core versus ZnCdSe/ZnMgS core/shell. The ZnSe coresappear to have an average size of about 5 nm, and the ZnCdSe core with aZnMgS shell has a diameter of between 8 and 9 nm, so the shell adds 3-4nm to the diameter of the nanocrystal.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are methods and materials that provide an effectiveshell layer on a Zn-containing nanocrystal core, which has beendifficult to achieve until now, and increases photostability of thenanocrystal. Without being bound by theory, the disclosed methods andmaterials apparently achieve increased photostability by providing ashell with a suitable bandgap to extend symmetrically beyond the bandgapof a Zn-containing nanocrystal core such as Zn_(x)Cd_(1-x)Se where x isat least about 0.25 or at least about 0.5, for example, and thusinsulate the core from the external environment on both the high and lowends of the core's bandgap.

In one embodiment (exemplified in FIGS. 8 and 9), a nanocrystal shellcomprises MgS or MgSe and a shell material such as ZnS, ZnSe. or CdSetogether. The combination of MgS with a second shell material such asMgSe or ZnS provides a bandgap that extends symmetrically both above andbelow the bandgap for a ZnSe core more effectively than either one alonewould, as illustrated in FIG. 5 for MgS plus ZnS. The increasedphotostability of the nanocrystals having magnesium included in theshell, which is seen in FIGS. 10-12, may be the result of this match ofthe bandgap of the shell with the core. Presumably the bandgap for themixture of MgS plus ZnS that is illustrated in FIG. 5 will have ahigher-end bandgap value that is higher than that of ZnS because it isincreased by the presence of MgS; the lower endpoint is similar for bothmaterials, so the mixture has little effect at that end of the bandgap.The bandgap analysis suggests that MgS alone would give a suitableshell, and it has been shown experimentally that the combination of MgSwith ZnS works very well.

The MgS and ZnS (or CdSe or ZnSe) may be present as two individual shelllayers in either order, or as a single layer containing Mg and Zn (orCd) mixed together. If present in two layers, the layers may be placedon the core in either order, i.e., the MgS layer may be the inner of thetwo layers, or the ZnS layer may be the inner of the two layers. Ifmixed together in one layer (see FIG. 9), the MgS and second shellmaterial such as ZnS may be mixed in any ratio that provides a bandgapwith improved properties over ZnS alone. If present in one layer, thelayer may be relatively homogeneous or it may occur as a gradient wherethe inner part of the layer is mainly ZnS or another suitable shellmaterial, and the ratio of MgS increases upon moving outward from thecore. Alternatively, if present in one layer, the layer may have ahigher Mg to Zn (or Cd) ratio nearest the core and the ratio maydecrease upon moving outward from the core.

Also disclosed herein are methods and compositions that providenanocrystals having greater luminescent intensity, or brightness, thanconventional nanocrystals that have CdSe cores. Brightness of ananocrystal determines how easily detected it will be in a system thatuses nanocrystals as labels or markers, thus increasing brightnessenhances the usefulness of a nanocrystal. The brightness that can beachieved by a nanocrystal is largely determined by its core, since thecore determines both the absorption characteristics and the emissioncharacteristics (quantum yield) that the nanocrystal can achieve. A corecomprising significant amounts of Zn is advantageous relative toconventional CdSe cores, because it increases brightness because itsabsorption of light in the visible spectrum tends to be better than thatof CdSe and it also provides high quantum yield. Accordingly, in someembodiments, the invention provides nanoparticles having a core thatcomprises Zn, as further described herein. In addition, a core thatcomprises Zn typically has a wider bandgap than a CdSe core, so it has

The brightness of a nanocrystal, however, can be lost rapidly if thecore is not protected from the environment by a shell or other coating.A coating or shell is needed to prevent the core from reacting with itsenvironment, particularly when it is in its photoactivated state. Thephotoactivated state has a ‘separated’ electron and hole, which canreact with species such as oxygen in ways that would not occur when thecore material was in its ground state. Such reactions degrade theluminescent properties of the core, but can be prevented by a suitablecoating or shell. In some embodiments, the invention thus provides acore having a shell, where the shell slows photodegradation of the core.In some embodiments, the shell is a magnesium-containing shell thatslows degradation relative to a similar shell lacking magnesium. Inaddition, the methods and compositions of the invention provide brighternanocrystals that emit in the green, blue and yellow parts of thevisible light spectrum, which complements the conventional CdSe corenanocrystals, which tend to emit in the red and orange region of thespectrum. Conventional nanocrystals do not provide blue or green colorshaving very high levels of luminescent intensity: nanocrystals havinggreater brightness in these emission regions are needed. Nanocrystals ofthe invention having blue, green or blue-green emission bands andbrightness that is significantly enhanced relative to conventional CdSecore nanocrystals are therefore an aspect of the invention.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein. “a” or “an” means “at least one” or “one or more.”

As used herein, ‘about’ means that the numerical value is approximateand small variations would not significantly affect the practice of theinvention. Where a numerical limitation is used, unless indicatedotherwise by the context, ‘about’ means the numerical value can vary by±10% and remain within the scope of the invention.

“Alkyl” as used in reference to alkyl phosphine, alkyl phosphine oxide,or alkylamine refers to a hydrocarbon group having 1 to 20 carbon atoms,frequently between 4 and 15 carbon atoms, or between 6 and 12 carbonatoms, and which can be composed of straight chains, cyclics, branchedchains, or mixtures of these. The alkyl phosphine, alkyl phosphineoxide, or alkylamine include embodiments having from one to three alkylgroups on each phosphorus or nitrogen atom. In preferred embodiments,the alkyl phosphine or alkyl phosphine oxide has three alkyl groups onP, and the alkyl amine(s) have one alkyl group on N. In someembodiments, the alkyl group contains an oxygen atom in place of onecarbon of a C4-C15 or a C6-C12 alkyl group, provided the oxygen atom isnot attached to P or N of the alkyl phosphine, alkyl phosphine oxide, oralkylamine. In some embodiments, the alkyl can be substituted by 1-3substituents selected from halo and C1-C4 alkoxy. Preferred alkylphosphines include compounds of the formula [(C₄-C₁₀)₃]P. Preferredalkyl phosphine oxides include compounds of the formula [(C₄-C₁₀)₃]P═O.Preferred alkylamines include compounds of formula (C₄-C₁₀)₂NH and(C₄-C₁₀)₂NH₂, where each C₄-C₁₀ alkyl is a straight or branched chainunsubstituted alkyl group. Preferred alkyl phosphonic acids and alkylphosphinic acids include those having 1-15 carbon atoms and preferably2-10 carbon atoms or 3-8 carbon atoms.

“Hydrophobic” as used herein refers to a surface property of a solid, ora bulk property of a liquid, where the solid or liquid exhibits greatermiscibility or solubility in a low-dielectric medium than it does in ahigher dielectric medium. A nanocrystal that is soluble in organicsolvents that are not miscible with water, such as ethyl acetate,dichloromethane, MTBE, hexane, or ether, is hydrophobic. By way ofexample only, nanocrystals that are soluble in a hydrocarbon solventsuch as decane and are insoluble in an alcohol such as methanol arehydrophobic.

“Hydrophilic” as used herein refers to a surface property of a solid, ora bulk property of a liquid, where the solid or liquid exhibits greatermiscibility or solubility in a high-dielectric medium than it does in alower dielectric medium. By way of example, a material that is moresoluble in methanol than in a hydrocarbon solvent such as decane wouldbe considered hydrophilic.

“Growth medium” as used herein refers to a mixture of reagents and/orsolvents in which a nanocrystals is grown or in which a shell is grownon a nanocrystals. These growth media are well known in the art, andoften include at least one metal, at least one chalcogenide (a compoundof S, Se, or Te), and one or more alkyl phosphines, alkyl phosphineoxides, alkyl phosphonic acids, alkyl phosphinic acids or alkylamines.

“Luminescence” refers to the property of emitting electromagneticradiation from an object. Typically, the electromagnetic radiation is inthe range of UV to IR radiation and may refer to visible electromagneticradiation, for example light. Luminescence may result when a systemundergoes a transition from an excited state to a lower energy stateresulting in the release of a photon. The transition responsible forluminescence can be stimulated through the release of energy stored inthe system chemically or kinetically, or can be added to the system froman external source, such as, for example by a photon or a chemical,thermal, electrical, magnetic, electromagnetic, physical energy source,or any other type of energy source capable of exciting the system. Insome embodiments, ‘luminescence’ refers to fluorescence-emission of aphoton that is initiated by excitation with a photon of higher energy(shorter wavelength) than the emitted photon.

“Exciting a system” or “exciting” or “excitation” refers to inducing theenergy state of a system into a higher state than that of ground state.The term “excitation wavelength” refers to electromagnetic energy whichmay have a shorter wavelength than that of the emission wavelength thatis used to excite the system. The “energy states” of the systemdescribed herein can be electronic, vibrational, rotational, or anycombination thereof. The term “emission peak” refers to the wavelengththat has the highest relative intensity within a characteristic emissionspectra.

The term “solid solution”, as used herein, may refer to a compositionalvariation that is the result of the replacement of an ion or ionic groupfor another ion or ionic group, for example, CdS in which some of the Cdatoms have been replaced with Zn. In contrast, a “mixture” or “alloy”,as used herein, may refer to a class of matter composed of two or moresubstances in which each substance retains its own identifyingproperties.

The term “monodisperse” refers to a population of particles havingsubstantially identical size and shape. For the purpose of the presentinvention, a “monodisperse” population of particles means that at leastabout 60% of the particles or, in some cases, about 75% to about 90% ormore of the particles, fall within a specific particle size range, andthe particles deviate in diameter or largest dimension by less than 10%rms (root-mean-square) and, in some cases, less than 5% rms. One ofordinary skill in the art will realize that particular sizes ofnanocrystals, such as of semiconductor nanocrystals, are actuallyobtained as particle size distributions.

While compositions and methods are described in terms of “comprising”various components or steps (interpreted as meaning “including, but notlimited to”), the compositions and methods can also “consist essentiallyof” or “consist of” the various components and steps, such terminologyshould be interpreted as defining essentially closed-member groups.

Nanocrystals

Any suitable nanocrystal can be used with the methods and themagnesium-containing shell coatings disclosed herein. The methods andshells of the invention provide increased photostability fornanocrystals. For example, the methods of the invention can be used tomodify the shell of a core-shell nanocrystal to increase itsphotostability. In some embodiments, the methods increase thephotostability relative to that of the same nanocrystal that has notbeen treated with magnesium so that the nanocrystal loses less thanabout 100/o of its emission intensity under photodegradation conditionsthat reduce the emission intensity of the untreated nanocrystal by 20%or by 40%. In some embodiments, the nanocrystals of the inventionprovide fluorescence intensity that is at least about 100% greater thanthat of a corresponding CdSe-core nanocrystal having the same emissionmaximum wavelength.

The methods of the invention are suitable for increasing the bandgap ofa shell comprising ZnS, for example, on any nanocrystal core: adding Mgto the ZnS of a shell increases the bandgap of the shell, thusincreasing its ability to shield the core. This increases photostabilityof the nanocrystal. The magnesium-containing shells of the invention aresuitable for use on any nanocrystal core having a bandgap with bothendpoints in the range between about −4 eV and −7 eV. A suitable exampleis a ZnSe or ZnCdSe nanocrystal core. The bandgap for CdSe suggests thatit should also work-see FIG. 2. ZnSe is another exemplary suitable shellmaterial.

Generally, a nanocrystal is a semiconductive particle, having a diameteror largest dimension in the range of about 1 nm to about 1000 nm, or inthe range of about 2 nm to about 50 nm, and in certain embodiments, inthe range of about 2 nm to about 20 nm. The term “semiconductornanocrystal” or “quantum dot” are used interchangeably herein to referto nanocrystals composed of a crystalline inorganic semiconductormaterial or mixture of materials. These nanocrystals, or quantum dots,are luminescent and are useful as fluorescent markers or labels thatfacilitate identifying, locating, tracking, or quantifying, etc.molecules, particles, cells and the like. A nanocrystal or quantum dotmay include one or more shells or other coatings in addition to asemiconductor core.

The nanocrystal is comprised of a core and a shell. The terms“semiconductive core”, “nanocrystal core”, “core nanocrystal” or “core”refer to a nanocrystal composed of an inorganic semiconductive material,a mixture or solid solution of inorganic semiconductive materials, or anorganic semiconductive material. Cores can be isolated, or can bepartially or fully covered by a shell or shells.

The term “shell” refers to an inorganic semiconductive layer surroundinga nanocrystal core. An “inorganic shell”, as described herein, is ashell composed of an inorganic material, or a mixture or solid solutionof inorganic materials. A suitable shell provides insulating abilityboth above and below the bandgap of the core material. A suitable shellfor a particular nanocrystal core will have a bandgap that is wider thanthe bandgap of the core, and that extends above the high end of thebandgap of the core and below the low end of the bandgap of the core. Incertain embodiments, the inorganic shell may be composed of aninsulating material or another semiconductive material. In someembodiments, the shell comprises magnesium and is referred to herein asa magnesium-containing shell. Some embodiments of the nanoparticles ofthe invention have a shell that comprises MgS. In some embodiments, theshell comprises or consists essentially of a mixture of ZnS and MgS. Insome embodiments, the mixture is chosen to provide a bandgap that ismore effective to provide photostabilization than a correspondingmagnesium-free semiconductive material.

“Core/shell nanocrystal” or “core/shell quantum dot” as used hereinrefers to a nanocrystal that includes a nanocrystal core of one or morefirst semiconductor materials contained within or substantially coatedwith a shell of a second inorganic material that is frequently asemiconductor, also.

Provided herein are semiconductor nanocrystals comprising a core and amagnesium-containing layer. The semiconductor nanocrystals can comprisemore than one magnesium-containing layer, such as 2, 3, 4, 5, 6, or morelayers. The core can be a semiconductor core. The semiconductor core cangenerally comprise any suitable semiconductor material, or mixtures oftwo or more suitable semiconductor materials. The core of asemiconductor nanocrystal can be a semiconductor material including, butnot limited to, those of Groups II-VI of the periodic table of elements,for example ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS,MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and thelike, and those of Groups III-V of the periodic table of elements, suchas GaN, GaP, GaAs, GaSb, and the like, and those of Group IV of theperiodic table of elements (Ge, Si, and the like), as well as alloys orcombinations or mixtures thereof. An example of a suitable semiconductorcore material is ZnX, where X is Se, S, or Te. Particular core materialsthat are well suited for the present invention as indicated by analysesof their bulk bandgaps and lattice spacing (mismatch of less than about4%) include ZnS, AlP, GaP, ZnSe, AlAs, GaAs, CdS, HgS, ZnSe and ZnCdSeand mixtures of any two of these. In certain embodiments, the corecomprises, consists essentially of, or consists of ZnSe and/or ZnCdSe ora mixture of these two.

Some particular embodiments of the core comprise Zn. Zn is advantageousbecause it provides nanocrystals that are larger than correspondingCd-based nanocrystals that emit at a particular fluorescence wavelength.Where Zn is used as a component of the core, it may be mixed with asecond metal such as Cd, Ga, Hg. or Al. Cd can be added, for example, toshift the color of the fluorescence toward green. The ratio of Zn toother metals if present is typically at least 0.25 or at least 0.5 or atleast about 1. In some embodiments the ratio is at least about 2.

In some embodiments, the core comprises or consists essentially of ZnSe.In some embodiments, the core comprises or consists essentially of amixture of ZnSe and CdSe in a ratio that provides a luminescentintensity that is at least about 50% higher than and optionally at leastabout 100% higher than the luminescent intensity of a CdSe nanocrystalcore having the same fluorescence emission maximum wavelength.Preferably, the ratio of Zn to Cd in a nanocrystal core for the presentinvention is at least 0.5 and frequently it is 1, or higher than 1, orhigher than about 2. In some embodiments, sufficient Cd is added toshift the emission wavelength to a wavelength above 500 nm.

FIG. 13 demonstrates how increasing the amount of Cd material used inpreparation of a ZnCdSe core increases the wavelength of the nanocrystalfluorescence to a wavelength between 510 and 520 nm, or between 515 and520 nm, or between 530 and 550 nm. Thus in some embodiments, theinvention provides a nanocrystal having a luminescent intensity that isat least about 50% higher than and optionally at least about 100% higherthan the luminescent intensity of a CdSe nanocrystal core having thesame fluorescence emission maximum wavelength, where the fluorescencewavelength is above 500 nm, or above 510 nm, or above 520 nm, or above530 nm, or above 540 nm, or between any two of these values, or to awavelength between about 510 and 550 nm.

The magnesium-containing layer can be a monolayer, or can be a thickerlayer comprised of several monolayers. The magnesium-containing layercan generally comprise any magnesium material. An example of a magnesiummaterial is MgX, where X is B. Al, Ga, In, C, Si, Ge, Sn, Pb, N, P, As,Sb, Bi, O, S, Se, or Te or mixtures. The semiconductor nanocrystals canfurther comprise a shell made of a second semiconductor materialdifferent from the core semiconductor material. The Mg-containing layercan be anywhere relative to the shell, but is preferable outside of theshell. The magnesium-containing layer may, in some embodiments, furthercomprise at least one additional semiconductive material. For example,the layer may be a homogeneous mixture of the magnesium material and theat least one semiconductive material, a gradient of the magnesiummaterial and the at least one semiconductive material, or layers of themagnesium material and the at least one semiconductive material.Gradients may have higher concentrations of MgX closest to the core, ormay have lower concentrations of MgX closest to the core.

In some embodiments, the semiconductor nanocrystal has a substantiallyuniform magnesium-containing layer. The uniformity can be determined byZ-contrast scanning transmission electron microscopy (Z-STEM), seeMcBride, et al., Nano Letters (2006) 6:1496-1501. In another embodiment,the magnesium-containing layer comprises at least 1, 2, 3, 4, 5, 6, ormore monolayers surrounding the entire core/shell of the nanocrystal.

Semiconductive material that may be used in the shell or shells ofcore/shell nanocrystals may include, but are not limited to, materialsmade up of a first element from Groups 2, 12, 13 or 14 of the PeriodicTable of the Elements and a second element from Group 16, for example,ZnS. ZnSe, ZnTe. CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe. MgTe, CaS,CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, and BaTe; materials made up of afirst element from Group 13 of the Periodic Table of the Elements and asecond element from Group 15, for example, GaN, GaP, GaAs, GaSb, InN,InP, InAs, InSb, and BP; materials made up of a Group 14 element, forexample. Ge and Si; materials such as PbS and PbSe; and alloys, solidsolutions, and mixtures thereof. In some embodiments, the shellcomprises Mg, or it comprises a mixture of Mg and Zn. In certainembodiments, the shell comprises MgS. Optionally, it can include MgSmixed with ZnS. The ratio of MgS to ZnS can vary from about 0.05 toabout 0.95, frequently it is between 0.1 and 0.8 and preferably it isbetween about 0.1 and about 0.5.

In some embodiments, the nanocrystals of the invention include two shelllayers. In certain embodiments, the first shell layer is selected fromCdSe, CdS, ZnS, and ZnSe and the second shell layer is selected from MgSand MgSe. The two shell layers may be disposed on the core in eitherorder. Thus embodiments of the invention include nanoparticles having acore of ZnSe or ZnCdSe with two shells; one of the shells comprisesCdSe, CdS, ZnS or ZnSe and the other shell comprises MgS or MgSe. Thetwo shells may be separate layers, but typically at least some blendingof the materials will occur at the interface between the two layers.Where the magnesium-containing layer contains S and a second shellmaterial is present, the second shell material may also comprise S.e.g., it can be ZnS or CdS. Where the magnesium layer comprises Se and asecond shell layer is present, the second shell material frequentlycomprises Se, e.g., it can be ZnSe or CdSe.

In other embodiments, the shell or shells may contain an additive whichmay be incorporated into the shell with one or more of the shellprecursors and may or may not be a semiconductive material. In someembodiments, the additive may be an element of Groups 2, 12, 13, 14, 15and 16, as well as Fe, Nb, Cr, Mn, Co, Cu, and Ni, and in others, theadditive may simply be a super-abundance of one of the core or shellprecursors. The additive may be incorporated into one or more layer ofthe shell, the core, or both the core and one or more layers of theshell, or may be present only in an interfacial region between the coreand the shell or between two layers of a multilayer shell. Theinterfacial region wherein the semiconductive core and shell meet maycontain elements of both the shell and core and of the additive. Whilenot wishing to be bound by theory, incorporating an additive into atleast the interfacial region of the nanocrystal may reduce stresses inthe core-shell interface caused by the differences in the latticestructures of the core and shell. Reduction of these stresses may serveto improve the strength and uniformity of the core-shell composite. Whenpresent in the shell, the additive may be uniformly distributedthroughout the shell or may be distributed as a gradient. For example,the additive may be present as a gradient that exhibits a decreasingconcentration in an outward direction from the semiconductive core. Incertain embodiments, the additive may not be incorporated into thenanocrystal at all, but may merely facilitate overgrowth of ahigh-quality thick shell on a core.

In still other embodiments, the shell may additionally contain one ormore materials that are not a semiconductive material, such as forexample, oxygen or other material present during preparation of theshell. These materials may be deliberately added to the reactionmixture, or they may occur adventitiously; commonly they do notmaterially affect the semiconductive properties of the shell material.

The shell may be made up of about 1 to about 20 monolayers and may,typically, be made up of about 4 to about 15 monolayers. Examples of thenumber of monolayers include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, and ranges between any two of these values.The diameter of a core/shell nanocrystal prepared using methods ofembodiments of the invention may be from about 1 nm to about 1000 nm, insome embodiments, about 2 nm to about 50 nm, and in certain embodiments,about 2 nm to about 20 nm. Specific examples of diameters include about2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm,about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about19 nm, about 20 nm, and ranges between any two of these values.

The nanocrystals may generally emit at any wavelength, or at any colorif the emission is in the visible spectrum. In some embodiments, thenanocrystals are blue or green, emitting at wavelengths of from about400 nm to about 600 nm, and in certain embodiments, the nanocrystals mayemit at below about 500 nm. Without wishing to be bound by theory,nanocrystals having a ZnX core may be better suited for makingnanocrystals that are blue or green emitting than the CdSe/ZnSnanocrystals currently used. Nanocrystals having a ZnX core that emitlight in the blue or green will be larger than nanocrystals having aCdSe core. Since larger cores have larger extinction coefficients, thismeans that ZnX cores that provide a particular emission wavelength willbe brighter and therefore more useful in applications where highsensitivity is required. “Brightness” as used herein refers to thefluorescence intensity provided by a nanocrystal. A “bright nanocrystal”is a nanocrystal that is not a conventional CdSe core/ZnSe shellnanocrystal, and which provides a greater fluorescence intensity than aconventional CdSe core/ZnSe shell nanocrystal having the samefluorescence emission wavelength and approximately the same shell layerthickness as the ‘bright nanocrystal.’ Qdot 525 is an example of aconventional nanocrystal, having a fluorescence emission wavelength of525 nm, and is used for some comparisons herein. For the purpose ofcomparing properties like this, two nanocrystals are viewed as havingthe ‘same’ emission wavelength if their emission maxima differ by lessthan about 5 nm.

The band gap energy of MgS may span the band gap energy of ZnSe orZnCdSe more symmetrically than ZnS. Without wishing to be bound bytheory, at least one monolayer of MgS coating a ZnSe or ZnCdSe core mayprovide coating which sufficiently insulates the core from the outsideenvironment, protecting the core electrons and holes from thedeleterious effects of such elements as oxygen and providing improvedchemical and photo stability. In addition, a mixture of MgS and ZnS mayprovide broader protection than MgS alone, as the mixture may provide abandgap that better covers and extends beyond that of ZnSe or ZnCdSe onboth the high and low ends of the core's bandgap. As illustrated inFIGS. 6 and 7, the emissions intensity of ZnCdSeZnS coreshellnanocrystals that have been treated with a magnesium reagent tointroduce MgS show improved stability over untreated ZnCdSe/ZnSnanocrystals.

Thus provided here are methods to improve the photostability of ananocrystal. Photostability can be assessed by methods described herein,by measuring the integrated emission intensity of the nanoparticle overtime under constant illumination. The illumination source was a 375 nmemitting Shark Series Visible LED purchased from Opto Technology, Inc.running at a constant current of 48 mA. A material is considered to bephotostable if it loses little of its fluorescent intensity duringirradiation for a 3 minute period under these conditions. In someembodiments, the nanocrystal is considered to be photostable if itretains 95% or more of its initial fluorescent intensity after thistreatment. In other embodiments, a nanocrystal is considered photostableif it retains at least 90% of its initial fluorescent intensity. Inother embodiments, a nanocrystal is considered photostable if it retainsat least 80% of its initial fluorescent intensity. In other embodiments,a nanocrystal is considered photostable if it retains at least 90% ofits initial fluorescent intensity under conditions where a reference,such as a nanocrystal that has not been treated with magnesium when usedas a reference for a nanocrystal that has been treated with magnesium,loses at least 20% of its initial emission intensity, or at least 40% ofits initial fluorescent intensity. FIGS. 6 and 7 depict tests ofphotostability done using this method. For this purpose, if hydrophobicnanoparticles were tested, then hexane was used to dilute the sample,and if hydrophilic nanoparticles were tested, then 50 mM borate bufferwas used to dilute the sample. These measurements are typically done tocompare the rate of loss of emission intensity of a nanocrystal to astandard; where a suitable standard is not otherwise disclosed, the corealone of a core/shell nanocrystal can be used as a standard, and testconditions can be adjusted so that the core loses at least about 40% ofits fluorescent intensity during the time course of the test. The testmaterial and standard should be tested at equivalent optical densities.In samples where the core alone is used as a reference standard,illumination can be maintained for a time period that causes thereference standard to lose 40% of its initial intensity, and thecore/shell nanocrystal is considered to be photostable relative to itscore when it loses less than about 10% of its fluorescent intensityduring the time required for the core alone to lose 40% of itsfluorescent intensity.

The methods of the invention are especially applicable to nanocrystalshaving a shell that comprises ZnS, and include treating the core/shellnanocrystal with a magnesium reagent under conditions that cause themagnesium to exchange into the shell to an extent that modifies thebandgap of the semiconductor material of the shell. In some embodiments,the method provides a nanocrystal having increased photostability.Photostability can be increased sufficiently for the treated nanocrystalto lose less than 10% of its fluorescence intensity under conditionsthat reduce the intensity of the untreated nanocrystal by 20%. In someembodiments, photostability is increased sufficiently for the treatednanocrystal to lose less than 10% of its fluorescence intensity underconditions that reduce the intensity of the untreated nanocrystal by40%.

One specific embodiment of the invention is a semiconductor nanocrystalcomprising a Zn_(x)Cd_(1-x)Se core, a ZnS first shell, and a MgS secondshell. The value “x” can be any number or fraction from 0 to 1. Thus thesemiconductor core comprises Zn and Cd in a ratio. In some embodiments,the ratio of Zn to Cd is at least 0.5. In other embodiments the ratio ofZn to Cd is at least 1 or at least about 2. In these embodiments, theZnS and MgS shells may be discrete, or they may intermix to any degree.The core contacts the first shell. The first shell contacts the core andthe second shell. The second shell contacts the first shell. This isgraphically shown in FIG. 8. Optionally, the order of the two layers canbe reversed, so the MgS layer is in contact with the core.

In some embodiments, provided herein is a method to increasefluorescence intensity by at least 100% over the fluorescence of aconventional nanocrystal having a CdSe core and the same fluorescenceemission maximum wavelength. In some embodiments, the method canincrease fluorescence intensity by at least 100% over the fluorescenceof a conventional nanocrystal having a CdSe core and the samefluorescence emission maximum wavelength. In these embodiments, theinvention also provides methods to maintain photostability of thenanocrystal. The methods are particularly useful to prepare nanocrystalsthat have a characteristic emission above about 500 nm, and providesnanocrystals having substantially superior brightness (emissionintensity) than conventional CdSe core nanocrystals like Qdot 525, acommercial standard, as demonstrated by FIGS. 10-12. Nanocrystals of theinvention thus include nanocrystals having photostability and alsohaving characteristic emission maxima above 500 nm, optionally between500 and 550 nm, while providing fluorescence intensity that is at leasttwice that of a corresponding CdSe core nanocrystal.

An additional specific embodiment of the invention is a semiconductornanocrystal comprising a Zn_(x)Cd_(1-x)Se core, and a Mg_(y)Zn_(1-y)Sfirst shell. The value “x” and “y” can be any number or fraction from 0to 1. In some embodiments y is selected by determining what amount of Mgis needed to provide at least a monolayer of MgS over the nanocrystal onwhich the shell is used, which the person of ordinary skill can readilycalculate from the size or surface area of the nanocrystal. The corecontacts the first shell. This is graphically shown in FIG. 9. Thecomposition of the core can vary across the range described above for ananocrystal core comprising a mixture of Zn and Cd together. Thecomposition of the shell can vary across the range disclosed above for ashell that comprises a mixture of Mg and Zn together. The result is ananocrystal having increased fluorescent intensity relative to ananocrystal with the same maximal fluorescence emission wavelengthhaving a CdSe core, and photostability comparable to that ofconventional nanocrystals having a CdSe core. While applicable tonanocrystals with fluorescence colors across the visible spectrum, thegreatest benefits of the invention may be achieved by the correspondingnanocrystals that have fluorescence maxima in the blue, green and yellowportions of the spectrum.

Methods of Preparation

Provided herein are methods for making nanocrystals disclosed herewithand methods for modifying known core/shell nanocrystals using theprinciples disclosed herein to increase the photostability of thenanocrystals. Alternative embodiments include various methods for thepreparation of semiconductor nanocrystals comprising a core and amagnesium-containing layer, as well as nanocrystals made by thedisclosed methods.

A method for the preparation of semiconductor nanocrystals usingsequential coating comprising a core and a magnesium-containing layercan comprise: providing a semiconductor core; coating the core with aninorganic shell to provide a core-shell intermediate; and contacting theintermediate with a magnesium material to produce a semiconductornanocrystal comprising a core and a magnesium-containing layer. The corecan generally be any semiconductor core, for example, ZnX, where X isSe, S, or Te.

The step of contacting the core/shell nanocrystal with the magnesiummaterial can be conducted under conditions similar to those describedherein. For example, the coreshell nanocrystal can be dispersed in asuitable liquid medium and contacted with the magnesium material ofchoice to form a mixture. The mixture can then be heated for anysuitable time period, usually between about 0.5 hr and 4 hr, and at anysuitable temperature, often between about 120° C. and about 250° C.

The coating step can comprise contacting the core with a first precursorand a second precursor. The first precursor can comprise a first elementfrom Groups 2, 12, 13 or 14 of the Periodic Table of the Elements. Thesecond precursor can comprise a second element from Group 16. Examplesof the inorganic shell can include, for example, ZnS, ZnSe, ZnTe, CdS,CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS,SrSe, SrTe, BaS, BaSe, and BaTe. Alternatively, the first precursor cancomprise a first element from Group 13 of the Periodic Table of theElements. The second precursor can comprise a second element from Group15. Examples of the inorganic shell can include, for example, GaN, GaP,GaAs, GaSb, InN, InP, InAs, InSb, and BP; materials made up of a Group14 element, for example, Ge and Si; materials such as PbS and PbSe; andalloys, solid solutions, and mixtures thereof.

The magnesium material can generally be any magnesium-containingmaterial. Examples of magnesium materials include Me₂Mg,dibutylmagnesium, MgO, MgF₂, MgCl₂, MgBr₂, MgI₂ Mg(acetoacetonate)₂,Mg(C₂H₃O₂)₂, MgTiO₃, MgWO₄, MgZrO₃, Mg₂Si, Mg(MnO₄)₂, Mg(ClO₄)₂, MgCO₃,MgB₂, bis(cyclopentadienyl)Mg, MgS₂O₃, MgSO₄, MgHPO₄, Mg(HSO₃)₂,MgAO₂O₄, and Mg(NO₃)₂. In some embodiments, the magnesium material foruse in the methods for introducing magnesium into a shell is selectedfrom magnesium bromide, magnesium acetate or another magnesiumalkylcarboxylate, and magnesium chloride; preferably the magnesiummaterial is magnesium acetate.

The magnesium-containing layer may be made up of about 1 to about 20monolayers and may, typically, be made up of about 4 to about 15monolayers. Examples of the number of monolayers include 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and rangesbetween any two of these values. The magnesium-containing layer can befrom less than 1 nm to more than 5 nm in thickness, for example it canbe from about 1 to about 5 nm in thickness or from about 2 to about 4 nmin thickness.

The contacting step with the magnesium material can generally beperformed under any suitable conditions and can be performed in thepresence of at least one solvent. Suitable solvents include fatty acidssuch as stearic acid or lauric acids; amines such as alkylamines ordodecylamine; phosphines such as trioctylphosphine; phosphine oxidessuch as trioctylphosphine oxide; phosphonic acids such astetradecylphosphonic acid; phosphoramides; phosphates; phosphates; andmixtures thereof. Other solvents including, for example, alkanes,alkenes, halo-alkanes, ethers, alcohols, ketones, esters, and the like,may also be useful in this regard, particularly in the presence of addednanocrystal ligands. It is to be understood that the first and secondsolvents may be the same solvent and, in “one-pot” type synthesis, maybe the same solution.

The contacting step can generally be performed at any suitabletemperature. For example, the temperature can be greater than about 120°C. or greater than about 150° C. or greater than about 200° C. Thetemperature can be held steady during the contacting step, or can bevaried during the contacting step.

This method offers certain advantages over methods that directly coat ananocrystal core with a magnesium-containing layer. For example, thismethod permits the user to produce a core/shell nanocrystal by knownmethods, which ensures that no lattice matching problems arise in thecoating step. While the known core/shell nanocrystals of this type haveless desirable photostability characteristics, the methods of theinvention provide a method to improve photostability and thus enhancethe usefulness of the nanocrystals significantly. These methods areparticularly well suited for use with a Zn-containing core, preferably acore that contains at least about 5% Zn. The methods can be used withcores such as ZnSe and ZnCdSe, as further described herein. Specificshells that are suitable as the shell of the core/shell nanocrystals inthese methods include those known to provide a stable shell on thesenanocrystal cores, such as ZnS and ZnCdS.

An alternative method for the preparation of semiconductor nanocrystalscomprising a core and a magnesium-containing layer coats thesemiconductor core and can comprise: providing a semiconductor core; andcontacting the semiconductor core with a magnesium material to produce asemiconductor nanocrystal comprising a core and a magnesium-containinglayer. The core can generally be any semiconductor core, for example,ZnX, where X is Se, S, or Te.

The magnesium material can generally be any magnesium-containingmaterial. Examples of magnesium materials include Me₂Mg,dibutylmagnesium, MgO, MgF₂, MgCl₂, MgBr₂, MgI₂ Mg(acetoacetonate)₂,Mg(C₂H₃O₂)₂ (magnesium acetate), magnesium formate, magnesiumpropionate, or other carboxylate salts of magnesium such as (RCO₂)₂Mgwhere R is alkyl, magnesium trifluoroacetate, magnesium benzoate, andthe like, MgTiO₃, MgWO₄, MgZrO₃, Mg₂Si, Mg(MnO₄)₂, Mg(ClO₄)₂, MgCO₃,MgB₂, bis(cyclopentadienyl)Mg, MgS₂O₃, MgSO₄, MgHPO₄, Mg(HSO₃)₂,MgAl₂O₄, and Mg(NO₃)₂. An alkylcarboxylate salt of magnesium or amixture of alkylcarboxylate salts of magnesium are used in certainembodiments.

The magnesium-containing layer may be made up of about 1 to about 20monolayers and may, typically, be made up of about 4 to about 15monolayers. Examples of the number of monolayers include 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and rangesbetween any two of these values.

The contacting step with the magnesium material can generally beperformed under any suitable conditions and the contacting step can beperformed in the presence of at least one solvent. Suitable solventsinclude fatty acids such as stearic acid or lauric acids; amines such asalkylamines or dodecylamine; phosphines such as trioctylphosphine;phosphine oxides such as trioctylphosphine oxide; phosphonic acids suchas tetradecylphosphonic acid; phosphoramides; phosphates; phosphates;and mixtures thereof. Other solvents including, for example, alkanes,alkenes, halo-alkanes, ethers, alcohols, ketones, esters, and the like,may also be useful in this regard, particularly in the presence of addednanocrystal ligands. It is to be understood that the first and secondsolvents may be the same solvent and, in “one-pot” type synthesis, maybe the same solution.

The contacting step can generally be performed at any suitabletemperature. For example, the temperature can be greater than about 150°C. or greater than about 200° C. The temperature can be held steadyduring the contacting step, or can be varied during the contacting step.

Yet another alternative method for the preparation of semiconductornanocrystals coats the core-shell nanocrystal and comprises a core and amagnesium-containing layer can comprise: providing a semiconductorcore-shell nanocrystal; and contacting the semiconductor core-shellnanocrystal with a magnesium material to produce a semiconductornanocrystal comprising a core and a magnesium-containing layer.

The core-shell nanocrystal can generally have any semiconductor core,for example, ZnX, where X is Se, S, or Te. The core-shell nanocrystalcan generally have any semiconductor shell, for example, ZnS, ZnSe,ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe,CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs, GaSb, InN, InP,InAs, InSb, BP, Ge, Si, PbS, PbSe, mixtures thereof, or alloys thereof.

The magnesium material can generally be any magnesium-containingmaterial. Examples of magnesium materials include Me₂Mg,dibutylmagnesium, MgO, MgF₂, MgCl₂. MgBr₂, MgI₂ Mg(acetoacetonate)₂,Mg(C₂H₃O₂)₂, MgTiO₃, MgWO₄, MgZrO₃, Mg₂Si, Mg(MnO₄)₂, Mg(ClO₄)₂, MgCO₃,MgB₂, bis(cyclopentadienyl)Mg, MgS₂O₃, MgSO₄. MgHPO₄, Mg(HSO₃)₂,MgAl₂O₄, and Mg(NO₃)₂.

The magnesium-containing layer may be made up of about 1 to about 20monolayers and may, typically, be made up of about 4 to about 15monolayers. Examples of the number of monolayers include 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and rangesbetween any two of these values.

The contacting step with the magnesium material can generally beperformed under any suitable conditions and the contacting step can beperformed in the presence of at least one solvent. Suitable solventsinclude fatty acids such as stearic acid or lauric acids; amines such asalkylamines or dodecylamine; phosphines such as trioctylphosphine;phosphine oxides such as trioctylphosphine oxide; phosphonic acids suchas tetradecylphosphonic acid; phosphoramides; phosphates; phosphates;and mixtures thereof. Other solvents including, for example, alkanes,alkenes, halo-alkanes, ethers, alcohols, ketones, esters, and the like,may also be useful in this regard, particularly in the presence of addednanocrystal ligands. It is to be understood that the first and secondsolvents may be the same solvent and, in “one-pot” type synthesis, maybe the same solution.

The contacting step can generally be performed at any suitabletemperature. For example, the temperature can be greater than about 150°C. or greater than about 200° C. The temperature can be held steadyduring the contacting step, or can be varied during the contacting step.

Additions to Methods

The above described methods may further comprise one or more steps ordetails. The following paragraphs describe exemplary additions to themethods.

For example, the method may include heating a solution containing ZnSecore nanocrystals in trioctylphosphine and admixing a Zn containingshell precursor, such as for example, Et₂Zn and a S containingprecursor, such as, for example. (TMSi)₂S both in trioctylphosphine tothe core mixture. A temperature sufficient to begin deposition of theshell onto the core may be maintained for a period of time. Thetemperature may then be decreased, and a Mg containing shell precursor,such as, for example, MgAc₂ (magnesium acetate) may be added to themixture. This mixture may then be heated and cooled allowingincorporation of the Mg containing precursor into the shell. In suchembodiments, a monolayer of MgS may be deposited as the outermost layerof the shell. Alternatively, the Mg containing precursor may beinitially added, or added prior to, or in addition to, the Zn or Scontaining shell precursor.

Examples of embodiments in which the shell contains an additive include,admixing the core-solvent mixture with an additive-first shell precursormixture made up of one or more additive(s) and the first shell precursorto form a first reaction mixture. The second shell precursor may besubsequently added to the first reaction mixture to form a secondreaction mixture. Heating to a temperature sufficient to induce shellformation may occur prior to, following, or concurrently with theaddition of additive-first shell precursor mixture to the core-solventmixture, or occur prior to, following, or concurrently with the additionof the second shell precursor to the first reaction mixture. In otherexamples, the core-solvent mixture may be admixed with a first shellprecursor to form a reaction mixture, and this reaction mixture may beadmixed with an additive-second shell precursor mixture containing oneor more additive and the second shell precursor. As in the aboveembodiments, this mixture may be heated to a temperature sufficient toinduce shell formation prior to, following, or concurrently with theaddition of additive-second shell precursor mixture to the core,solvent, first shell precursor mixture.

The method of embodiments of the invention may be carried out in asingle reaction vessel, a “one-pot” synthesis, or may be carried outusing separate syntheses for the semiconductive core and the inorganicshell. Particle size and particle size distribution during the growthstage of the core and shell may be approximated by monitoring theabsorption or emission peak positions and line widths of the samples,and dynamic modification of reaction parameters such as temperature andmonomer concentration in response to changes in the spectra allows thetuning of these characteristics.

Core nanocrystals may be prepared by many methods. In some embodiments,first and second core precursors may be injected into a reactionsolution that may be held at a temperature sufficient to inducehomogeneous nucleation of discrete particles. In some embodiments, oneor more additive may also be added to the reaction solution used forcore preparation. The particles may then be allowed to grow until thedesired size has been reached, such as, for example, a diameter of fromabout 20 {acute over (Å)} (2 nm) to about 125 {acute over (Å)} (12.5nm). In some embodiments, the growth process may be stopped by droppingthe reaction temperature.

Cores thus prepared may be isolated using methods known to those skilledin the art, such as, for example, flocculation with a non-solvent, forexample, methanol. In some embodiments, a monodisperse particlepopulation containing the individual cores may be obtained. The coresmay be isolated and/or purified, using methods known to those skilled inthe art, from a first solvent and then placed in a second solvent toform a core solution, or in some embodiments, the solution containing amonodisperse nanocrystal population can be used “as is”, meaning thatfurther purification or isolation of the cores may not be necessary oncecore synthesis is completed.

In some embodiments, nanocrystals may be covered with an organic orother overcoating on an outer surface of the nanocrystal. Theovercoating may be made up of a material capable of providingcompatibility with a suspension medium and a moiety possessing anaffinity for the outermost surface of the nanocrystal. For example,suitable overcoating materials may include, but are not limited to,polystyrene, polyacrylate, or other polymers, such as polyimide,polyacrylamide, polyethylene, polyvinyl, poly-diacetylene,polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone,polypyrrole, polyimidazole, polythiophene, and polyether; epoxies;silica glass; silica gel; titania; siloxane; polyphosphate; hydrogel;agarose; cellulose; and the like. These overcoating materials may attachdirectly to the outermost surface of the nanocrystal or may beassociated with a moiety known to intimately associate with the surfaceof nanoctystals such as, for example, a mercapto, phosphine, orimidazole moiety and the like. The coating may be in the range of about2 to about 100 nm thick in some embodiments and about 2 to about 10 nmin others.

Methods of Use

The compositions provided herein are particularly beneficial inapplication where enhanced sensitivity is required. For example, whenperforming enzyme-linked immunosorbent assays (ELISA), the brightness ofthe fluorescently labeled antibody is directly related to the overallsensitivity of the assay. For conventional CdSe based core/shell labeledantibodies, red colors are far more sensitive than green, for example.This can make multiplexed assays difficult if the concentrations ofantigens are unknown because it is difficult to provide a wide range ofreadily-distinguished nanocrystal colors while maintaining sufficientsensitivity to detect very low concentrations. FIGS. 10 and 11 show howcompositions provided in this invention provide higher emissionintensities relative to commercially available CdSe based core-shellnanocrystals.

Additionally, the compositions provided herein are beneficial influorescent cell labeling applications where high sensitivity isrequired. FIG. 12 shows the relative intensities of nanocrystals of oneembodiment of the disclosed compositions compared to conventional,commercially available antibody conjugated nanocrystals that emits lightat 525 nm. The data show that the composition provided here hassignificantly higher emission intensities relative to conventionalnanocrystals.

The following examples are included to demonstrate some embodiments ofthe disclosed methods and compositions. It should be appreciated bythose of skill in the art that the techniques disclosed in the exampleswhich follow represent techniques discovered by the inventor(s) tofunction well in the practice of the invention, and thus can beconsidered to constitute preferred modes for its practice. However,those of skill in the art should, in light of the present disclosure,appreciate that many changes can be made in the specific embodimentswhich are disclosed and still obtain a like or similar result withoutdeparting from the scope of the invention.

EXAMPLES Example 1: Preparation of Luminescent ZnSe Nanocrystal Cores

To a 50 mL round bottom reaction flask under inert atmosphere, 15 mL ofmolten hexadecylamine, 98% and 47 μL of Bis (2,4,4,-trimethylpentyl)phosphinic acid (CYANEX 272 from Cytec Industries Inc) were added. Themixture was heated to 100° C. under vacuum while stirring and held for15 minutes. After the 15 minute hold time, the flask was refilled withnitrogen and further heated to 310° C. At 310° C., a mixture of 0.123 g(1.0 mmol) diethylzinc (Et₂Zn) and 1.274 g of 1 M TOP-Se (1.4 mmol Se)(TOP-Se was prepared by dissolving selenium metal in trioctylphosphine)was swiftly injected into the flask. After a 15 minute hold time, anadditional syringe of 0.370 g Et₂Zn (3.0 mmol of Zn) and 3.822 g of 1 MTOP-Se (4.2 mmol of Se) was prepared and added at a rate of 18.5μL/minute for 15 minutes, 50 μL/minute for 30 minutes and 75 μL/minutefor 15 minutes. When the precursor addition was completed, the reactionwas cooled to room temperature. The final material had an emissionmaximum wavelength of 424 nm and a full width at half maximum (FWHM) of15 nm. The peak wavelength of the first absorbance feature was 413 nm.The optical density at 413 nm was 34.4.

Example 2: Preparation of Luminescent ZnSe Nanocrystal Cores

To a 50 mL round bottom reaction flask under inert atmosphere, 15 mL ofmolten hexadecylamine (HDA), 98% was added. The HDA was heated to 100°C. under vacuum while stirring and held for 15 minutes. After the 15minute hold time, the flask was refilled with nitrogen and furtherheated to 310° C. At 310° C. a mixture of 0.123 g (1.0 mmol) diethylzinc(Et₂Zn) and 1.274 g of 1 M TOP-Se (1.4 mmol Se) (TOP-Se was prepared bydissolving selenium metal in trioctylphosphine) was swiftly injectedinto the flask. After a 15 minute hold time, an additional syringe of0.370 g Et₂Zn (3.0 mmol of Zn) and 3.822 g of 1 M TOP-Se (4.2 mmol ofSe) was prepared and added at a rate of 18.5 ML/minute for 15 minutes,50 μL/minute for 30 minutes and 75 μL/minute for 15 minutes. When theprecursor addition was completed, the reaction was cooled to roomtemperature. The final material had an emission maximum wavelength of413 nm and a FWHM of 19 nm. The peak wavelength of the first absorbancefeature was 408 nm. The optical density at 408 nm was 63.3. The averagediameter as measured by TEM for fifty particles was 5.2 nm with astandard deviation of 0.7 nm.

Example 3: Preparation of Luminescent ZnCdSe Nanocrystal Cores

To any given volume of molten ZnSe cores as prepared in Example 1, 6volumes of toluene followed by 2 volumes of butanol were added. Afterthoroughly mixing this mixture, 7 volumes of methanol were added. Thecloudy mixture was centrifuged at about 3000 rpm for 10 minutes. Thesupernatant was discarded and 1 volume of hexane was added to dissolvethe nanocrystal-containing pellet. The concentration of the hexanesolution was determined by measuring the absorbance at the firstabsorbance feature and dividing by the extinction coefficient. Theextinction coefficient was calculated with the following equation, wherethe particle diameter was measured by TEM.

ε=4.8080×(particle diameter in angstroms)³

To a 50 mL round bottom reaction flask under inert atmosphere, 6.3 mL ofmolten hexadecylamine and 163 nmoles of ZnSe cores in hexane (fromabove) were added. The flask was held under vacuum until all volatilesolvents were removed. Next, 597 μL of a mixture of 1.072 gdimethylcadmium (Me₂Cd), 3.57 mL of CYANEX 272, and 25.89 mLtrioctylphosphine is added (149 μmol of Cd). Different amounts ofcadmium solution have been employed to give other emission wavelengths.Several examples of the effects of cadmium addition on the finalemission wavelength of the cores/shells are provided in FIG. 13.

This mixture was heated to 255° C., and held at temperature for 20minutes. If a core-shell was the final desired product, the shellprecursors are immediately added after the 20 minutes hold time (seeExample 6). Otherwise, the flask was allowed to cool to roomtemperature. The final material had an emission maximum wavelength of498 nm and a FWHM 40 nm.

Example 4: Preparation of Luminescent ZnCdSe Nanocrystal Cores

To any given volume of molten ZnSe cores as prepared in Example 2, 6volumes of toluene followed by 2 volumes of butanol were added. Afterthoroughly mixing this mixture, 7 volumes of methanol were added. Thecloudy mixture was centrifuged at about 3000 rpm for 10 minutes. Thesupernatant was discarded and 1 volume of hexane was added to dissolvethe nanocrystal-containing pellet. The concentration of the hexanesolution was determined by measuring the absorbance at the firstabsorbance feature and dividing by the extinction coefficient. Theextinction coefficient was calculated with the following equation, wherethe particle diameter was measured by TEM.

ε=4.8080×(particle diameter in angstroms)³

To a 50 mL round bottom reaction flask under inert atmosphere, 6.3 mL ofmolten hexadecylamine and 150 nmoles of ZnSe cores in hexane (fromabove) were added. The flask was held under vacuum until all volatilesolvents were removed. Next, 301 μL of a mixture of 0.108 gdimethylcadmium (Me₂Cd), 0.358 mL of CYANEX 272, and 2.602 mLtrioctylphosphine is added (75.3 μmol of Cd).

This mixture was heated to 255° C., and held at temperature for 20minutes. If a core-shell was the final desired product, the shellprecursors are immediately added after the 20 minutes hold time (seeExample 7). Otherwise, the flask was allowed to cool to roomtemperature. The final material had an emission maximum wavelength of495 nm and a FWHM 40 nm.

Example 5: Coating with Zinc Sulfide Shell

To a volume of molten ZnSe cores as prepared in Example 1, 6 volumes oftoluene, followed by 2 volumes of butanol were added. After thoroughlymixing this mixture, 7 volumes of methanol were added. The cloudymixture was centrifuged at about 3000 rpm. The supernatant was discardedand 1 volume of hexane was added to dissolve the pellet. Theconcentration of the hexane solution was determined by measuring theabsorbance at the first absorbance feature and dividing by theextinction coefficient. The extinction coefficient was calculated withthe following equation, where the particle diameter was measured by TEM.

ε=4.8080×(particle diameter in angstroms)³

To a 50 mL round bottom reaction flask under inert atmosphere, 6.3 mL ofmolten hexadecylamine and 150 nmoles of ZnSe cores in hexane (fromabove) were added. The flask was held under vacuum until all volatilesolvents were removed. This mixture was heated to 255° C. When thesolution reached 255° C., 2.2 mL of a mixture of 0.186 g Et₂Zn, 0.717 mLCYANEX 272, and 5.155 mL trioctylphosphine (0.55 mmol Zn), and 1.1 mL amixture of 0.269 g bis(trimethylsilyl)sulfide ((TMS)₂S) and 2.243 gtrioctylphosphine (0.55 mmol S) were added sequentially over 87 minutes.The number and duration of the sequences were determined by calculatingthe moles of zinc and sulfur atoms required to deliver four monolayersof ZnS; each 3 angstroms (0.3 nm) thick. Each sequence started withzinc. When precursor addition was completed, the mixture was cooled to100° C. and 9.8 mL of toluene was added. After the toluene addition, themixture was allowed to cool to room temperature. The final material hadan emission maximum wavelength of 422 nm and a FWHM 19 nm. The quantumyield of this material in organic solvent was measured in a mixture of25% TOP and 75% 2-methyl-THF. TOP is a good oxygen scavenger and isrequired when measuring the quantum yield of oxygen sensitive materials.The quantum yield in the above organic solvent was 31%. When thematerial was redispersed in a mixture of 95% 50 mM borate solution and5% 2-mercaptoethanol with a hydrophobically modified hydrophilicpolymer, the quantum yield was 20% (Example 11). The water dispersibleparticles dispersed in a solution of 50 mM borate lost 75% of theirinitial emission intensity after 3 min of constant illumination. Theaverage diameter as measured by TEM for fifty particles was 8.3 nm witha standard deviation of 1 nm.

Example 6: Coating with Zinc Sulfide Shell

Immediately following the 20 minute hold time at the end of the Example3, 1.922 mL of a mixture of 0.925 g Et₂Zn, 3.57 mL CYANEX 272 and 25.65mL trioctylphosphine (0.48 mmol Zn), and 0.961 mL of a mixture of 0.128g (TMS)₂S and 1.072 g trioctylphosphine (0.48 mmol of S) are addedsequentially over 96 minutes. The number and duration of the sequenceswere determined by calculating the moles of zinc and sulfur atomsrequired to deliver four monolayers of ZnS each 3 angstroms (0.3 nm)thick. Each sequence started with zinc. When precursor addition wascompleted, the mixture was cooled to 100° C. and 9.6 mL of toluene wasadded. After toluene addition, the mixture was allowed to cool to roomtemperature. The final material had an emission maximum wavelength of525 nm and a FWHM 44 nm.

Example 7: Coating with Zinc Sulfide Shell

Immediately following the 20 minute hold time at the end of the Example4, 2.2 mL of a mixture of 0.167 g Et₂Zn, 0.645 mL CYANEX 272 and 4.639mL trioctylphosphine (0.55 mmol Zn), was mixed with 0.22 mL of a mixtureof 0.269 g Me₂Cd(0.055 mmol Cd), 0.896 mL of CYANEX 272 and 6.505 mL oftrioctylphosphine and added sequentially with 1.1 mL of a mixture of0.269 g (TMS)₂S and 2.243 g trioctylphosphine (0.55 mmol of S) over 96minutes. The number and duration of the sequences were determined bycalculating the moles of zinc and sulfur atoms required to deliver fourmonolayers of ZnS each 3 angstroms (0.3 nm) thick. Each sequence startedwith zinc. When precursor addition was completed, the mixture was cooledto 100° C. and 9.8 mL of toluene was added. After toluene addition, themixture was allowed to cool to room temperature. The final material hadan emission maximum wavelength of 519 nm and a FWHM 48 nm. The organicquantum yield in hexane was 47%. The quantum yield of the particlesdispersed in 50 mM borate with a hydrophobically modified hydrophilicpolymer was 42% (Example 11). The organic particles dispersed in hexanelost 40% of their initial emission intensity after 3 min of constantillumination (FIG. 6). The water dispersible particles in a solution of50 mM borate lost 28% of their initial emission intensity after 3 min ofconstant illumination (FIG. 7).

Example 8: Treatment with Magnesium

To a 50 mL round-bottom flask, 857.8 mg magnesium acetate hydrate, 5.07mL CYANEX 272, and 14.34 mL trioctylphosphine were added. Under asweeping flow of nitrogen, the mixture was heated to 250° C. thenimmediately cooled back to room temperature. Next, 4 mL of isopropylalcohol and 12 mL of methanol were added to 15.9 mL of core-shells fromExample 6. The cloudy solution was thoroughly mixed and centrifuged at3000 rpm for 10 minutes. The supernatant was discarded and 15.9 mL ofhexane was added to redissolve the pellet. White insoluble solids wereremoved by centrifugation to yield a clear, colored dispersion ofnanocrystals. The particles were further precipitated as before andredispersed in the magnesium solution from above. Once the pellet wascompletely dispersed in the magnesium solution, the mixture was degassedat room temperature under vacuum. Finally, the mixture was heated to180° C. and held there for 1 hour. After the 1 hour hold time, themixture was cooled to room temperature. The final material had anemission maximum wavelength of 520 nm and a FWHM 41 nm. The organicquantum yield in hexane was 59%. The quantum yield of the particlesdispersed in 50 mM borate with a hydrophobically modified hydrophilicpolymer was 50% (Example 11).

Example 9: Treatment with Magnesium

To a 50 mL round-bottom flask, 646.5 mg magnesium acetate hydrate, 3.82mL CYANEX 272, and 10.81 mL trioctylphosphine were added. Under asweeping flow of nitrogen, the mixture was heated to 250° C. thenimmediately cooled back to room temperature. Next, 2.5 mL of isopropylalcohol and 7.5 mL of methanol were added to 10 mL of core-shells fromExample 7. The cloudy solution was thoroughly mixed and centrifuged at3000 rpm for 10 minutes. The supernatant was discarded and 10 mL ofhexane was added to redissolve the pellet. White insoluble solids wereremoved by centrifugation to yield a clear, colored dispersion ofnanocrystals. The particles were further precipitated as before andredispersed in the magnesium solution from above. Once the pellet wascompletely dispersed in the magnesium solution, the mixture was degassedat room temperature under vacuum. Finally, the mixture was heated to180° C. and held there for 1 hour. After the 1 hour hold time, themixture was cooled to room temperature. The final material had anemission maximum wavelength of 515 nm and a FWHM 39 nm. The quantumyield of the organic particles dispersed in hexane was 67%. When thematerial was redispersed in 50 mM borate solution with a hydrophobicallymodified hydrophilic polymer, the quantum yield was 51%. The organicparticles dispersed in hexane lost 4% of their initial emissionintensity after 3 min of constant illumination (FIG. 6). The waterdispersible particles dispersed in a solution of 50 mM borate lost 4% oftheir initial emission intensity after 3 min of constant illumination(FIG. 7). The average diameter as measured by TEM for fifty particleswas 8.5 nm with a standard deviation of 0.9 nm. FIG. 14 shows acomparison of particle sizes for the ZnSe cores from Example 2 andZnCdSe/ZnMgS core/shells prepared from this example.

Example 10: Preparation of Hydrophobically Modified Hydrophilic Polymersfor Attachment to Nanocrystal Surface

Hydrophobically modified hydrophilic polymers were prepared as describedin U.S. Pat. No. 6,649,138 to Adams et. al., as follows.

A modified polyacrylic acid was prepared by diluting 100 g [0.48 molCOONa] of poly(acrylic acid. sodium salt) (obtained from Aldrich,molecular weight 1200) two-fold in water and acidifying in a 1.0 L roundbottom flask with 150 ml (1.9 mol) of concentrated HCl. The acidifiedpolymer solution was concentrated to dryness on a rotary evaporator (100mbar, 80° C.). The dry polymer was evacuated for 12 hours at less than10 mbar to ensure water removal. A stirbar and 47.0 g (0.24 mol) of1-[3-(dimethyl-amino)-propyl]-ethylcarbodiimide hydrochloride(EDC-Aldrich 98%) were added to the flask, and the flask was then sealedand purged with N₂, and fit with a balloon. 500 ml of anhydrousN,N-dimethylformamide (Aldrich) was transferred under positive pressurethrough a cannula to this mixture; and the flask was swirled gently todissolve the solids. 32 ml (0.19 mol) of octylamine was transferreddropwise under positive pressure through a cannula from a sealedoven-dried graduated cylinder into the stirring polymer/EDC solution,and the stirring continued for 12 hours. This solution was concentratedto <100 ml on a rotary evaporator (30 mbar, 80° C.), and the polymer wasprecipitated by addition of 200 ml di-H₂O (de-ionized water) to thecooled concentrate, which produced a gummy white material. This materialwas separated from the supernatant and triturated with 100 ml di-H₂Othree more times. The product was dissolved into 400 ml ethyl acetate(Aldrich) with gentle heating, and basified with 200 ml di-H₂O and 100 gN—N—N—N-tetramethylammonium hydroxide pentahydrate (0.55 mol) for 12hours. The aqueous layer was removed and precipitated to a gummy whiteproduct with 400 ml of 1.27 M HCl. The product was decanted andtriturated with 100 ml of di-H₂O twice more, after which the aqueouswashings were back-extracted into 6×100 mL portions of ethyl acetate.These ethyl acetate solutions were added to the product flask, andconcentrated to dryness (100 mbar, 60° C.). The crude polymer wasdissolved in 300 mL of methanol and purified in two aliquots over LH-20(Amersham-Pharmacia-5.5 cm×60 cm column) at a 3 mL/minute flow rate.Fractions were tested by nmR for purity, and the pure fractions werepooled, while the impure fractions were re-purified on the LH-20 column.After pooling all of the pure fractions, the polymer solution wasconcentrated by rotary evaporation to dryness, and evacuated for 12hours at <10 mbar. The product was a white powder (25.5 g, 45% oftheoretical yield), which showed broad nmR peaks in CD₃OD [δ=3.1 b (94),2.3 b (9.7), 1.9 1.7 1.5 1.3 b (63.3) 0.9 bt (11.3)], and clear IRsignal for both carboxylic acid (1712 cm⁻¹) and amide groups (1626 cm⁻¹,1544 cm⁻¹).

Example 11: Preparation of Water-Dispersible Quantum Dots UsingHydrophobically Modified Hydrophilic Polymers

The following procedure was used to provide water-dispersiblenanocrystals from a number of the preceding examples. Hydrophobicquantum dots were dispersed in an aqueous solvent system usinghydrophobically modified hydrophilic polymers as described in U.S. Pat.No. 6,649,138, as follows.

Twenty milliliters of 3-5 μM (3-5 nmoles) of TOPO/TOP coated core-shellnanocrystals, prepared in Examples 5, 6, 7, 8, or 9, were precipitatedwith 20 milliliters of methanol. The flocculate was centrifuged at3000×g for 3 minutes to form a pellet of the nanocrystals. Thesupernatant was thereafter removed and 20 milliliters of methanol wasagain added to the particles. The particles were vortexed to looselydisperse the flocculate throughout the methanol. The flocculate wascentrifuged an additional time to form a pellet of the nanocrystals.This precipitation/centrifugation step was repeated an additional timeto remove any excess reactants remaining from the nanocrystal synthesis.Twenty milliliters of chloroform were added to the nanocrystal pellet toyield a freely dispersed sol.

300 milligrams of the hydrophobically modified poly(acrylic acid) wasdissolved in 20 mL of chloroform. Tetrabutylammonium hydroxide (1.0 M inmethanol) was added to the polymer solution to raise the solution to pH10 (pH was measured by spotting a small aliquot of the chloroformsolution on pH paper, evaporating the solvent and thereafter wetting thepH paper with distilled water). Thereafter the polymer solution wasadded to 20 mL of chloroform in a 250 mL round bottom flask equippedwith a stir bar. The solution was stirred for 1 minute to ensurecomplete admixture of the polymer solution. With continued stirring thewashed nanocrystal dispersion described above was added dropwise to thepolymer solution. The dispersion was then stirred for two minutes toensure complete mixing of the components and thereafter the chloroformwas removed in vacuo with low heat to yield a thin film of theparticle-polymer complex on the wall of the flask. Twenty milliliters ofdistilled water were added to the flask and swirled along the walls ofthe flask to aid in dispersing the particles in the aqueous medium. Thedispersion was then allowed to stir overnight at room temperature. Atthis point the nanocrystals were freely dispersed in the aqueous medium.

Example 12: Preparation of Polyethoxylated Modified Water DispersibleQuantum Dots

Water dispersible quantum dots from Example 11 are mixed with a 7000fold molar excess of polyethylene glycol (PEG) molecules. PEG moleculesor mixtures of PEG molecules ranging in molecular weight from 200 to20,000 Da and having various functionalities such as methoxy, hydroxyl,amine, carboxy, phosphate, thiol, azido, NHS ester, aldehyde,isocyanate, and biotin can be used. Next, a 3000 fold molar excess ofEDC was added and the mixture was stirred for two hours. Excess PEG andEDC were removed by ultrafiltration. The quantum yield ofpolyethoxylated, water dispersible nanocrystals from Example 8 was 37%.The quantum yield of polyethoxylated, water dispersible nanocrystalsfrom Example 9 was 50%.

Example 13: Preparation of Quantum Dot Streptavidin Conjugates

To polyethoxylated modified water dispersible quantum dots from Example12, a 100 fold molar excess of Bis(Sulfosuccinimidyl)suberate (BS³) wasadded and gently mix for 30 minutes. Excess BS³ was removed via purifiedover an Illustra™ NAP-5 column (GE Healthcare). Purified BS³ activatedparticles from the NAP-5 column were mixed with a 40 fold molar excessor streptavidin and allowed to incubate at room temperature for 2 hours.Remaining unreacted BS³ was quenched with a 1 M solution of glycine. Acertain volume of 1 M glycine solution was added to the streptavidinconjugated particles such that the solution contained 50 millimoles ofglycine per liter of solution. The conjugates were purified from excessglycine by ultrafiltration. The quantum yield of a streptavidinconjugate from core shell from Example 9 was 38%. The brightness ofthese streptavidin conjugates was compared to a commercially availableCdSe core based streptavidin conjugate in an ELISA assay (FIG. 10). Thestreptavidin conjugates provided by this invention were approximately4.6 fold brighter than the commercially available nanocrystals.

Example 14: Preparation of Quantum Dot Secondary Antibody Conjugates

To polyethoxylated modified water dispersible quantum dots from Example12, a 130 fold molar excess ofSuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) wasadded. This solution was gently mixed and incubated at room temperaturefor 1 hour. SMCC activated quantum dots were purified from excess SMCCover an illustra NAP-25 column. In a second reaction container, 0.35 mgof Fab or F(ab′)₂ antibody fragments per nanomole of polyethoxylatedparticles were mixed with 4000 nanomoles of Dithiothreitol (DTT) permilligram of antibody and gently mixed for 30 minutes. DTT reducedantibody was purified from excess DTT over an illustra NAP-25 column.Activated quantum dots and reduced antibody were mixed and allowed toincubate at room temperature for 2 hours. Remaining unreacted SMCC wasquenched by incubating conjugates for 30 minutes with a 45 fold molarexcess of 2-mercaptoethanol. Antibody conjugates were purified by fastprotein liquid chromatography (FPLC). The quantum yield of a goatanti-rabbit conjugate from a core-shell from example 8 was 31%. Thebrightness of these antibody conjugates was compared to a commerciallyavailable CdSe core based antibody conjugate in an ELISA assay (FIG.11). The antibody conjugates provided by this invention wereapproximately 4.5 fold brighter than the commercially availablenanocrystals. Additionally, the brightness of these antibody conjugateswas compared to a commercially available CdSe core based antibodyconjugate in a cellular labeling application (FIG. 12). The antibodyconjugates provided by this invention were approximately 2 to 3 foldbrighter than the commercially available nanocrystals depending on theexcitation wavelength.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from the conceptand scope of the invention. More specifically, it will be apparent thatcertain agents which are chemically related may be substituted for theagents described herein while the same or similar results would beachieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the scope and conceptof the invention.

Example 14: Relative Photostability Experimental Tests

Photostability experiments were performed on both hydrophobic andhydrophilic nanoparticles. If hydrophobic nanoparticles were tested,then hexane was used to dilute the sample and reference material and toadjust the optical densities to be equivalent. If hydrophilicnanoparticles were tested then 50 mM borate buffer was used to dilutethe sample and reference materials and to adjust the optical densitiesto be equivalent. A reference material was always used in thephotostability experiments. The reference materials were generallyeither a non-magnesium containing nanoparticle or a commercial CdSenanoparticle; where no other reference is identified, the core alone(with no shell) of a core/shell nanocrystal can be used as a referencestandard. The experiment was performed by measuring the integratedemission intensity of the nanoparticle over time under constantillumination. The illumination source was a 375 nm emitting Shark SeriesVisible LED purchased from Opto Technology, Inc. running at a constantcurrent of 48 mA.

1.-20. (canceled)
 21. A process for making a photostable nanocrystalcomprising the steps of: i) providing a first core/shell nanocrystal;ii) dispersing the nanocrystal in a reaction mixture containing amagnesium material; and iii) heating the reaction mixture with thenanocrystal to a temperature of at least about 120° C.; to provide aphotostable nanocrystal having better photostability than the firstcore/shell nanocrystal.
 22. The process of claim 21, wherein thephotostable nanocrystal loses less than 10% of its initial emissionintensity under conditions where the first core/shell nanocrystal wouldlose 20% of its initial emission intensity.
 23. The process of claim 21or claim 22, wherein the reaction mixture comprises an alkylphosphinicacid, a trialkylphosphine, or a trialkylphosphine oxide, or a mixture ofat least two of these materials.
 24. The process of claim 21, whereinthe photostable nanocrystal loses less than 10% of its initial emissionintensity under conditions where the first core/shell nanocrystal wouldlose 40% of its initial emission intensity.
 25. The process of claim 21,wherein the core of the first core/shell comprises ZnSe.
 26. The processof claim 21, wherein the core of the first core/shell comprises ZnCdSe.27. The process of claim 26, wherein the ratio of Zn to Cd is at leastabout
 1. 28. The process of claim 26, wherein the amount of Cd in thecore provides a core having a largest dimension of about 6 nm and afluorescence color in the blue or green region of the visible spectrum.29. The process of claim 26, wherein the amount of Cd in the coreprovides a core having a largest dimension of about 6 nm and afluorescence emission maximum at a wavelength above 500 nm.
 30. Theprocess of claim 21, wherein the shell of the first core/shellnanocrystal comprise ZnS.
 31. The process of claim 21, wherein themagnesium material is a magnesium alkylcarboxylate.
 32. The process ofclaim 31, wherein the magnesium alkylcarboxylate is magnesium acetate.33. The process of claim 21, wherein the reaction mixture comprises atleast one material selected from a trialkylphosphine, atrialkylphosphine oxide, an alkylamine, and an alkylphosphinic acid. 34.The process of claim 33, wherein the reaction mixture comprisestrioctylphosphine.
 35. The process of claim 21, wherein the step ofheating the reaction mixture with the nanocrystal comprises heating themixture to a temperature between about 150° C. and about 250° C.
 36. Theprocess of claim 35, wherein the step of heating the reaction mixturewith the nanocrystal comprises heating the mixture for between about 0.5hr and about 4 hr.
 37. A photostable nanocrystal made by the process ofclaim
 21. 38. A nanocrystal comprising a semiconductor core and a shell,wherein the shell comprises Mg.